The determination of the presence of an antigen at the surface of a cell by immunofluorescence is a well-known technique. Usually, such determination involves the use of antibodies of polyclonal or monoclonal origin which bind specifically to this antigen. The presence of this antigen is revealed directly by the antibody which has been labelled with a fluorescent tag, or indirectly by reacting a second antibody which binds the first antibody, this second antibody bearing the immunofluorescent tag. These well-known procedures might be simple to execute at the expense of a high level of sensitivity.
A more sensitive approach involves the use of complement in cytotoxicity tests, the complement having this particularity to bind the constant fragment (Fc) of an antibody, and to create pores in the cells bearing the immune complex. When the target cells are lymphocytes, this test is called lymphotoxicity test. Lymphotoxicity tests usually need purified lymphocytes to insure an enrichment of the preparation to be analyzed and/or to get rid of other cells, particularly of erythrocytes, which could be identified as lymphocytes, and counted as such.
Preparations of purified lymphocytes can be obtained after more or less laborious procedures, which implies that a good level of sensitivity is gained at the expense of the simplicity and rapidity of execution.
There is clearly a need for a method of typing lymphocytes, which would reconcile good sensitivity as well as rapidity of testing.
The human leucocyte antigen (HLA) system is now known as the major histocompatibility complex (MHC). The highly polymorphic genes of this complex encode proteins, that are commonly referred to as MHC molecules or HLA antigens. Expressed on the surface of a variety of cells, these proteins are the principal determinants of graft rejection. Also, they have a major role in immune response and serve as genetic markers associated with disease susceptibility.
One of the most important associations known is certainly the HLA-B.sub.27 with ankylosing spondylitis (AS) and reactive arthritis (RA). More than 95% of patients with AS and approximately 70% of the patients with RA bear HLA-B.sub.27 antigen. The prevalence of this gene product is around 7% in normal Caucasian, 7% in Chinese, 6% in Mexican, 4% in American black and 0.8% in Japanese populations. The HLA-B.sub.27 determination by a simple method could find a diagnostic use in conjunction with a clinical and radiological evidences.
Up to now, the most commonly known and used methods to evaluate the HLA-B.sub.27 determinant are classified in two categories.
The first category regroups plate tests sold by the following companies: Gen Track Inc., 5100, Campus Drive, Plymouth Meeting, Pa. 19462; One lambda Inc., 21001, Kittridge St., Canoga Park, Calif. 91303-2801; and Laboratoire Omega Ltee, 11177, Hamon, Montreal (Quebec), H3M 3E4. These tests are performed using plates coated with polyclonal antibodies and to which is added a test sample after purification of its leucocytes, and using the following general method:
isolating of lymphocytes on a Ficoll gradient, PA1 mixing the lymphocytes with antiserum contained in test plates, PA1 allowing for immune complexes to be formed during about 30 minutes, PA1 adding complement to these immune complexes and incubating for about 60 minutes, PA1 fixing and staining the lymphocytes with formaldehyde and eosin or trypan blue, and PA1 reading the results under an inverted phase-contrast microscope in the case eosin was used as a staining agent or under a microscope without phase contrast in the case trypan blue was used as a dye. PA1 the simultaneity of the reactions antibody-antigen, fixation of the complement and staining of the positive cells, PA1 the combination of cytotoxicity test with fluorescent staining, and PA1 no purification of the target cell necessary as far as blood cell population is considered.
Whatever dye is used, the lysed cells (positive results) are dark and swollen while the negative cells are well-stained and retain their morphological characteristics. When the rate of lysis is at least 30% higher than in negative control, the result is assessed positive.
The second category regroups diagnostic kits containing monoclonal antibodies sold by the following companies: Becton Dickinson, 2350, Qume Drive, San Jose, Calif. 95131-1807; and One Lambda (address given above), and involves classical immunofluorescence procedure. The antibody is a monoclonal antibody which has been labelled with a fluorochrome. Laborious preparation of the lymphocytes to type might be rendered necessary because any cell susceptible to bind the labelled Fc fragment, like macrophages, monocytes, neutrophils, B cells and natural killer cells, must be eliminated from the preparation before the latter is reacted with these antibodies, in order to avoid false positive results. The identification and numbering of positive cells are made by flow cytometry, which means that the accessibility of this typing is reduced to the institutions which possess such a costly apparatus. Furthermore, the test can be performed by a highly skilled technician trained to operate this apparatus. Moreover, the interpretation of the results is rendered difficult by the absence of easy-to-interpret negative and positive controls. This test is not suitable for direct visualization under fluorescence microscopy unless the population of cells to test is very rich in antigens.
Therefore, there is clearly a need for a simple and sensitive procedure which would be available to any small laboratory simply possessing facilities and apparatus like a low-speed centrifuge and a fluorescence microscope. The use of a fluorochrome like propidium iodide which is commonly used to stain cell DNA might be advantageously used instead of dyes like eosin and trypan blue, for a direct evaluation of the positive cells.